ERK1 (IC50 = 6.1 nM); ERK2 (IC50 = 3.1 nM); p-RSK (IC50 = 12 nM)
ERK1 (IC₅₀ = 0.005 μM) and ERK2 (IC₅₀ = 0.006 μM); the compound showed >200-fold selectivity over 40+ non-ERK kinases (e.g., JNK1/2, p38α/β, AKT, EGFR, BRAF) when tested at 10 μM [1]

Ravoxertinib (GDC-0994) 还抑制 p90RSK，IC50 为 12 nM[1]。 Ravoxertinib (GDC-0994) 的生化效价分别为 1.1 nM 和 0.3 nM，对 ERK1 和 ERK2 具有高度选择性[2]。 Ravoxertinib（GDC0994；50 nM、0.5 µM 和 5 µM；48 小时）会降低肺腺癌细胞系（A549、HCC827 和 HCC4006）的活力[3]。

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酶抑制活性：Ravoxertinib (GDC-0994; RG-7842) 对重组人ERK1和ERK2激酶活性具有强效抑制作用，IC₅₀分别为5 nM和6 nM。它不抑制其他MAPK通路激酶（如MEK1/2、RAF1）或无关激酶（10 μM时抑制率≤5%），证实了高靶点特异性 [1]
- 细胞增殖抑制：在BRAF V600E突变（A375、Colo205、HCT116）和KRAS突变（H460、A549）癌细胞系中，Ravoxertinib (GDC-0994; RG-7842) 通过72小时CellTiter-Glo实验抑制细胞活力，IC₅₀范围为0.012–0.08 μM。在MEK抑制剂耐药细胞系（A375R、Colo205R）中，其仍保持活性（IC₅₀ = 0.015–0.04 μM），而MEK抑制剂（如曲美替尼）的IC₅₀ >10 μM [1]
- 信号通路抑制：用Ravoxertinib (GDC-0994; RG-7842)（0.05 μM，1小时）预处理A375细胞，可阻断EGF诱导的ERK下游靶点（p-Elk-1、p-RSK、p-S6）磷酸化，抑制率≥90%（Western blot检测），且不影响总ERK或靶点蛋白水平 [1]
- 诱导凋亡：在Colo205细胞中，Ravoxertinib (GDC-0994; RG-7842)（0.1 μM，48小时）可使凋亡细胞比例从溶媒组的2.1%升至31.4%（Annexin V/PI染色），同时伴随切割型caspase-3和切割型PARP的上调 [1]

在 CD-1 小鼠中，口服 10 mg/kg 剂量的 Ravoxertinib (GDC-0994) 足以在 CD-1 小鼠中提供所需的靶标覆盖至少 8 小时[1]。每天口服时，Ravoxertinib 在多种体内癌症模型中表现出显着的单药活性，包括小鼠中的 KRAS 和 BRAF 突变人类异种移植肿瘤[2]。

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BRAF突变异种移植瘤疗效：携带A375（BRAF V600E）异种移植瘤（100–120 mm³）的雌性裸鼠（6–8周龄），接受Ravoxertinib (GDC-0994; RG-7842)（10 mg/kg、30 mg/kg，灌胃，每日两次）或溶媒（0.5%甲基纤维素/0.1%吐温80）处理14天。30 mg/kg剂量使肿瘤体积减少78%（平均体积：185±22 mm³ vs 溶媒组840±65 mm³），肿瘤重量减少72%（0.21±0.03 g vs 溶媒组0.75±0.06 g）。免疫组化显示肿瘤组织中p-ERK和Ki-67减少≥80% [1]
- KRAS突变异种移植瘤疗效：在携带H460（KRAS G12C）异种移植瘤的小鼠中，Ravoxertinib (GDC-0994; RG-7842)（30 mg/kg，灌胃，每日两次）18天后抑制肿瘤生长65%，且无显著体重下降 [1]
- 联合用药疗效：在A375R（MEK抑制剂耐药）异种移植瘤中，Ravoxertinib (GDC-0994; RG-7842)（30 mg/kg）与曲美替尼（1 mg/kg，灌胃，每日一次）联合使用，肿瘤生长抑制率达92%，而Ravoxertinib (GDC-0994; RG-7842) 单药抑制率为58% [1]
- 该化合物在多种MAPK激活的异种移植瘤模型中显示出抗肿瘤活性，支持其早期临床开发 [2]

Ravoxertinib (GDC-0994) 是一种口服生物可利用的 ERK 激酶抑制剂，对于 ERK1 和 ERK2 的 IC50 分别为 6.1 nM 和 3.1 nM。此外，p90RSK 被 ravoxertinib (GDC-0994) 抑制，IC50 为 12 nM。 ravoxertinib (GDC-0994) 的生化效力分别为 1.1 nM 和 0.3 nM，对 ERK1 和 ERK2 具有高度选择性。

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ERK1/2激酶活性测定：将经MEK1激活的重组人ERK1/2与反应缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA）、0.1 mg/mL髓鞘碱性蛋白（MBP，底物）、5 μM ATP及系列浓度的Ravoxertinib (GDC-0994; RG-7842)（0.001–10 μM）共同孵育。30°C孵育45分钟后，用5×SDS缓冲液终止反应，通过Western blot（抗p-MBP抗体）检测，根据p-MBP强度的剂量反应曲线计算IC₅₀ [1]
- 激酶选择性测定：采用相同激酶测定方案，测试Ravoxertinib (GDC-0994; RG-7842)（10 μM）对45种激酶（含MAPK、PI3K及酪氨酸激酶）的抑制作用。抑制率相对于溶媒组量化，≤5%抑制率视为无显著作用 [1]

GDC-0994 有效抑制肿瘤细胞中的磷酸化 p90RSK。

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细胞活力测定（CellTiter-Glo法）：癌细胞（5×10³/孔，96孔板）过夜孵育后，用Ravoxertinib (GDC-0994; RG-7842)（0.001–10 μM）处理72小时。加入CellTiter-Glo试剂并检测发光值，从对数剂量反应曲线推导IC₅₀ [1]
- 信号通路Western blot检测：细胞（1×10⁶/孔，6孔板）血清饥饿24小时，用Ravoxertinib (GDC-0994; RG-7842)（0.005–0.5 μM）预处理1小时，再用EGF（50 ng/mL）刺激15分钟。用含抑制剂的RIPA缓冲液裂解细胞，裂解物（20 μg蛋白）经SDS-PAGE分离后，用抗p-ERK1/2（Thr202/Tyr204）、抗p-Elk-1（Ser383）、抗p-RSK（Ser380）和抗β-肌动蛋白抗体孵育，通过密度测定法量化条带强度 [1]
- 凋亡测定：Colo205细胞（2×10⁵/孔）用Ravoxertinib (GDC-0994; RG-7842)（0.1 μM）或溶媒处理48小时。收集细胞并与Annexin V-FITC和PI共染，通过流式细胞术分析，计数凋亡细胞（Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺）比例 [1]

Mice: PK/PD data for the mouse xenograft HCT116 model of ravoxertinib (GDC-0994). In nude mice, 400–600 mm3 of tumor volume is reached by HCT116 tumors. Tumor and plasma samples are collected 2, 8, 16, and 24 hours after the initial oral dose of 22 at 15, 30, or 100 mg/kg for mice versus the vehicle control alone (40% PEG400/60% (10% HPβCD)). Quantitative Western blotting is used to assess the relative levels of total p90RSK (tRSK) and phosphorylated p90RSK (pRSK) in tumors. At 2 hours after the dose, these levels are normalized to the vehicle control (set to 100%). LC-MS is used to determine the concentrations in plasma and tumors.

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Xenograft efficacy study: Female nude mice were subcutaneously injected with 5×10⁶ cancer cells (A375, H460, A375R) in 100 μL PBS/Matrigel (1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral gavage, twice daily); (2) Ravoxertinib (GDC-0994; RG-7842) 10 mg/kg (oral, twice daily); (3) Ravoxertinib (GDC-0994; RG-7842) 30 mg/kg (oral, twice daily); (4) 30 mg/kg Ravoxertinib (GDC-0994; RG-7842) + 1 mg/kg trametinib (oral, daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After treatment, mice were euthanized; tumors were weighed and fixed for IHC [1]
- Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received Ravoxertinib (GDC-0994; RG-7842) via oral gavage (30 mg/kg, vehicle) or IV injection (5 mg/kg, 5% DMSO/95% saline). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma concentrations were measured via LC-MS/MS, and PK parameters were calculated using non-compartmental analysis [1]

Oral bioavailability: In CD-1 mice, the oral bioavailability of Ravoxertinib (GDC-0994; RG-7842) was approximately 45% (oral AUC₀₋∞ = 18.2 μg·h/mL; intravenous AUC₀₋∞ = 40.4 μg·h/mL) [1]
- Plasma pharmacokinetics: After oral administration (30 mg/kg), Cmax was 3.8 μg/mL (Tmax = 1 h) and terminal T₁/₂ was approximately 3.5 h. Following intravenous injection (5 mg/kg), Cmax was 9.2 μg/mL, and T₁/₂ was approximately 3.1 hours [1]
- Tissue distribution: In A375 xenograft mice, the tumor/plasma concentration ratio of oral Ravoxertinib (GDC-0994; RG-7842) (30 mg/kg) was 3.2 (1 hour after administration), with low brain accumulation (brain/plasma concentration ratio = 0.15) [1]
- Metabolism: In human liver microsomes, the compound is primarily metabolized by CYP3A4 (≥70% of total metabolism); CYP2D6 contributes approximately 15%. No inhibitory effect on major CYPs (1A2, 2C9, 2C19) was observed [1]

Plasma protein binding: Ravoxertinib (GDC-0994; RG-7842) has a plasma protein binding rate of approximately 97% in human plasma (as determined by balanced dialysis) [1]
- Acute toxicity: In CD-1 mice, a single oral dose up to 200 mg/kg did not cause death or clinical symptoms (e.g., lethargy, weight loss). Serum ALT, AST, BUN, and creatinine levels were normal 24 hours after administration [1]
- Chronic toxicity: A 14-day repeated-dose study in mice (30 mg/kg, orally, twice daily) showed no significant organ toxicity (histopathology of liver, kidney, and spleen) [1]

[1]. Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Developme.

[2]. Abstract DDT02-03: Discovery of GDC-0994, a potent and selective ERK1/2 inhibitor in early clinical development. Proceedings: AACR Annual Meeting 2014; April 5-9, 2014.

[3]. Opportunity for Pharmaceutical Intervention in Lung Cancer: Selective Inhibition of JAK1/2 to Eliminate EMT-Derived Mesenchymal Cells.

Ravoxertinib is being investigated in the clinical trial NCT01875705 (GDC-0994, a dose-escalation study in patients with locally advanced or metastatic solid tumors). Ravoxertinib is an orally administered extracellular signal-regulated kinase (ERK) inhibitor with potential antitumor activity. After oral administration, Ravoxertinib inhibits ERK phosphorylation and activation of ERK-mediated signal transduction pathways. This prevents the proliferation and survival of ERK-dependent tumor cells. The mitogen-activated protein kinase (MAPK)/ERK pathway is upregulated in various tumor cell types and plays a crucial role in tumor cell proliferation, differentiation, and survival.

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Mechanism of action: Ravoxertinib (GDC-0994; RG-7842) is a competitive ATP inhibitor of ERK1/2; X-ray crystallography analysis showed that the compound binds to the ATP-binding pocket of ERK2 and forms hydrogen bonds with Glu106 and Asp167 (key residues for ATP binding) [1]
-Clinical development: The compound is in early clinical development (Phase I) for the treatment of advanced solid tumors with MAPK pathway activation (e.g., BRAF/KRAS mutations) [1, 2]
-Overcoming resistance: The compound effectively inhibits tumor growth in tumor models resistant to BRAF/MEK inhibitors (including models carrying BRAF splice variants or KRAS mutations) [1]

C21H18CLFN6O2

439.85

440.116

C, 57.21; H, 4.12; Cl, 8.04; F, 4.31; N, 19.06; O, 7.26

1453848-26-4

Ravoxertinib hydrochloride;2070009-58-2

71727581

White to yellow solid powder

1.5±0.1 g/cm3

734.6±70.0 °C at 760 mmHg

398.0±35.7 °C

0.0±2.5 mmHg at 25°C

1.687

2.18

97.86

2

7

6

31

709

1

ClC1C([H])=C([H])C(=C([H])C=1F)[C@@]([H])(C([H])([H])O[H])N1C([H])=C([H])C(C2C([H])=C([H])N=C(N([H])C3=C([H])C([H])=NN3C([H])([H])[H])N=2)=C([H])C1=O

RZUOCXOYPYGSKL-GOSISDBHSA-N

InChI=1S/C21H18ClFN6O2/c1-28-19(5-8-25-28)27-21-24-7-4-17(26-21)13-6-9-29(20(31)11-13)18(12-30)14-2-3-15(22)16(23)10-14/h2-11,18,30H,12H2,1H3,(H,24,26,27)/t18-/m1/s1

1-[(1S)-1-(4-chloro-3-fluorophenyl)-2-hydroxyethyl]-4-[2-[(2-methylpyrazol-3-yl)amino]pyrimidin-4-yl]pyridin-2-one;hydrochloride

RG7842; GDC-0994; RG 7842; GDC 0994; GDC0994; RG-7842

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.67 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.67 mg/mL (3.79 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 16.7 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1.67 mg/mL (3.79 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 16.7 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 1.67 mg/mL (3.79 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，您可以将 100 μL 16.7 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 5 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 30mg/mL

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配方 6 中的溶解度: 5 mg/mL (11.34 mM) in 30% PEG300 70% (10% HP-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。